Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.
Trinkle-Mulcahy
Laura
L
Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, Scotland, UK. ltrinkle@uottawa.ca
Boulon
Séverine
S
Lam
Yun Wah
YW
Urcia
Roby
R
Boisvert
François-Michel
FM
Vandermoere
Franck
F
Morrice
Nick A
NA
Swift
Sam
S
Rothbauer
Ulrich
U
Leonhardt
Heinrich
H
Lamond
Angus
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D030562:Databases, Protein; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008863:Microspheres; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012685:Sepharose; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism
D046912:Multiprotein Complexes; D020543:Proteome; D011993:Recombinant Fusion Proteins; C506234:USP9X protein, human; D049452:Green Fluorescent Proteins; D012685:Sepharose; D043222:Ubiquitin Thiolesterase